Rama 25 is a clonal epithelial cell line derived from a dimethylbenzanthracene-induced rat mammary adenocarcinoma. In the presence of the mammotrophic hormones, insulin, hydrocortisone, estrogen and prolactin, Rama 25 produces small amounts of casein and forms domes at a low rate. The rates of both these processes can be greatly increased by the addition of dimethyl sulphoxide or hexamethylenebisacetamide which are also known to induce the differentiation of Friend erythroleukeniia cells. Other compounds which stimulate the differentiation of Rama 25 cells include linoleic acid and 6-thioguanine. The intracellular pathways triggering changes in the two markers of differentiation are partially separable using different combinations of hormones, prolactin and hydrocortisone being the most important for the production of casein and the formation of domes respectively. The kinetics of differentiation, as judged by the appearance of these two markers, are characterised by two phases, a fixed period of 8 h (lag phase), the length of which is independent of the dimethyl sulphoxide concentration and a second phase where their rates are dependent on the concentration of dimethyl sulphoxide. Rama 25 cells do not become comitted to differentiate during this lag phase but increasing numbers of cells do so after this period. We suggest that the differentiation processes occur in two stages. The first stage, involving the inducer, commits Rama 25 cells to a new differentiated state. The second stage, involving the hormones, modulates the expression of different markers of this state. Both casein production and dome formation can be blocked by inhibitors of DNA synthesis and show reciprocal changes with the rates of cellular DNA synthesis. Thus, in its hormonal and D N A synthetic requiremcnts for differentiation, Rama 25 cells appear to resemble some of the mammary epithelial cells of mature virgin rats.Mammary gland growth and differentiation in vivo is controlled by a series of interacting hormones, notably prolactin, estrogens, glucocorticoids, progesterone and insulin [l -41. These hormones apparently cause both growth and development of ducts into lobulo-alveolar structures [5]. The former process predominantly involves epithelial cell proliferation while the latter process requires both epithelial cell proliferation and differentiation into specialised alveolar milksecreting cells. During lactation, the alveolar cells synthesis and secrete a number of milk-specific substances, the major protein component being casein in the rat [6]. The mammotrophic hormones, insulin, hydrocortisone and prolactin, have also been observed to induce the synthesis ofcasein in vitro in both virgin and mid-pregnant mouse and rat mammary gland explants [7, 81 and in dispersed cultures of primary mammary epithelial cells [9, 101. Increases both in levels of casein mRNA and in the efficiency of its translation occur prior to the onset of the synthesis of casein during lactation [Ill. However, the cellular events following the interaction of th...

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A competition e.l.i.s.a. (enzyme-linked immunosorbent assay) is described that enables direct measurement of the muscle-specific polypeptide of chick creatine kinase (M-CK) in extracts of differentiating muscle-cell cultures and in blood plasma samples, even in the presence of embryonic, or brain-type, creatine kinase. The characteristics of the assay can be considerably improved by the use of a monoclonal antibody, CK-ART, instead of rabbit antisera, and we offer an explanation for this in terms of heterogeneity of antibody affinities in polyclonal antisera. In addition to native enzyme, the assay will measure creatine kinase unfolded and inactivated by 8 M-urea treatment. During chick muscle differentiation in vitro, M-CK increased from 7.5% of the total creatine kinase at 24h to 76.0% at 143h, in good agreement with isoenzyme separation data. As a percentage of the total cell protein, M-CK increased by 156-340-fold over the same period and constituted 0.38-0.56% of the total protein in late cultures. E.l.i.s.a. measurements on 17-20-day embryonic thigh-muscle extracts, which contain almost exclusively M-CK, agree well with enzyme activity and radioimmunoassay. M-CK constituted 0.7-1.6% of the total protein in 17-19-day embryonic thigh muscle. Plasma M-CK concentrations in normal 2-8-week-old chickens were found to be in the range 0.5-0.9 micrograms/ml. Plasma concentrations of 32-56 micrograms/ml were found in 8-week-old dystrophic chickens by both e.l.i.s.a. and enzyme-activity measurements. The results suggest that inactive or unfolded forms of M-CK do not normally exist, in any significant amounts, in cell and tissue extracts or in freshly prepared samples of plasma.

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